This invention pertains to sense-antisense nucleic acid complexes, particularly to sense-antisense nucleic acid complexes which are hyperstabilized with a selective binding agent.
Nucleic acids, which encode the genetic information of all cells and viruses, commonly occur in two forms, known as DNA and RNA. A chain of several bases of RNA or DNA is called an oligonucleotide or a polynucleotide, depending on its size. Two oligonucleotides or polynucleotides whose base sequences are complementary to one another can bind to one another into a double helix through a process called base-pairing.
So-called xe2x80x9cantisensexe2x80x9d oligonucleotides or probes have been investigated as a possible means for regulating gene expression. An antisense oligonucleotide is an oligonucleotide (usually a synthetic oligonucleotide introduced from an external source) whose sequence is complementary to that of a target sequence of DNA or RNA (usually RNA) of a cell or virus. Because the antisense probe is complementary to the target sequence, the two can bind into a double helix. This induced double helix inhibits the activity of the target sequence, at least temporarily. The inhibition can result from failure of the RNA-DNA heteroduplex to be recognized as a template for translation, or as a result of the activation of RNase H by the RNA-DNA duplex. RNase H hydrolyses RNA in an RNA/DNA duplex. RNase H occurs in some viruses including HIV, bacteria, and ubiquitously in plant and animal cells. Thus RNA bound in such an RNA/DNA heteroduplex can be inactivated either because it is not recognized as a single-stranded messenger RNA, or because it is hydrolyzed after the heteroduplex forms.
Antisense technology is expected to have important therapeutic applications directed toward a variety of diseases, possibly including cancer, viral and retroviral diseases, autoimmune diseases, and parasitic infections.
Some structurally modified antisense probes have also been tested for potential therapeutic activity. These modifications generally render them resistant to nucleases, enzymes which degrade nucleic acids. These modifications are most commonly made to the phosphodiester linkage connecting adjacent nucleotides, but some sugar analogs and xe2x80x9cartificialxe2x80x9d stereoisomers of sugar-base linkages have also been devised.
Unfortunately, the resulting increase in nuclease resistance has been offset by a reduction in the stability of the sense-antisense duplex. A more stable sense-antisense duplex would permit smaller doses of antisense oligonucleotides to be used with greater effect. Previous strategies to enhance the stability of sense-antisense duplexes have included the attachment of intercalating acridines or reactive agents such as alkylating agents or psoralens (which is photoactive), but these approaches have met with only limited success.
Prior antisense technologies relying on the exogenous application of antisense oligonucleotides have encountered problems with the stability of the antisense probe, with the uptake of the antisense probes by cells, and with the stability of the sense-antisense duplexes formed. The first of these limitations, stability of the probe, is believed to be due to the susceptibility of single-stranded nucleotides (with conventional phosphodiester or PO linkages) to digestion by nucleases present in serum and in cell culture medium. The second limitation, uptake of the probes by cells, is believed to be due to poor uptake of the polyanionic (i.e., electronically charged) oligonucleotide through the cell membrane, although some studies have suggested the existence of an xe2x80x9coligonucleotide pumpxe2x80x9d which may transport charged oligonucleotides. Modified internucleotide linkages, such as those using methyl phosphonate (MP) or phosphorothioate (PS) linkages, have partially overcome the first two limitations. These modifications can result in improved resistance to nucleases, and somewhat enhanced cell penetration. See Uhlmann et al., xe2x80x9cAntisense Oligonucleotides: A New Therapeutic Principle,xe2x80x9d Chemical Reviews, vol. 90, no. 4, pp. 543-584 (1990); and Cohen, xe2x80x9cOligonucleotides as Therapeutic Agents,xe2x80x9d Pharmac. Ther., vol. 52,pp. 211-225 (1991), the entire disclosures of both of which are incorporated by reference.
But treating the first two limitations has increased the difficulties caused by the third limitation, the problem of poor stability of sense-antisense hybrids (as measured by Tm, the strand separation temperature). With methylphosphonate antisense structures, an additional problem was also found: the antisense-sense duplexes did not stimulate RNase H.
Uhlmann et al., xe2x80x9cAntisense Oligonucleotides: A New Therapeutic Principle,xe2x80x9d Chemical Reviews, vol. 90, no. 4, pp. 543-584 (1990) review generally the field of antisense technology, including the use of modified backbones in the oligonucleotides; the tethering of intercalating agents and psoralen; effects on RNase H activation; and limitations and strengths of the prior antisense technology.
Hxc3xa9lxc3xa8ne, xe2x80x9cArtificial Control of Gene Expression by Oligodeoxynucleotides Covalently Linked to Intercalating Agents,xe2x80x9d Br. J. Cancer, vol. 60, pp. 157-160 (1989); and Lee et al., xe2x80x9cInteraction of Psoralen-Derivatized Oligodeoxyribonucleoside Methylphosphonates with Single-Stranded DNA,xe2x80x9d Biochemistry, vol. 27, no. 9, pp. 3197-3203 (1988) discuss the use of crosslinking agents (such as psoralens and alkylators) and acridines linked to antisense oligonucleotides, with limited increases observed in the resulting stabilization of the duplexes.
Cohen, xe2x80x9cOligonucleotides as Therapeutic Agents,xe2x80x9d Pharmac. Ther., vol. 52, pp. 211-225 (1991) discusses uses of oligonucleotides as therapeutic agents, including uses in triple-strand binding. Cohen noted that precious approaches to antisense technology have been more successful in preventing translation than in preventing elongation.
Reynolds et al., xe2x80x9cThe Chemistry, Mechanism of Action and Biological Properties of CC-1065, A Potent Antitumor Antibiotic,xe2x80x9d J. Antibiotics, vol. 39, No. 3, pp. 319-334 (1986) reviews the properties of CC-1065 and routes for its synthesis.